ProjectSummary ClostridiumdifficiletoxinB(TcdB)isamajorvirulentfactorresponsiblefordiseasesassociated with C. difficile infection. Our understanding of TcdB pathogenesis remains limited, owing in large part to a lack of established receptors through which to understand the in vivo targeting anddiseaseprogressionincolonictissues.Werecentlycarriedoutgenome-widescreensusing the CRISPR-Cas9 approach and identified two distinct candidate receptors: Frizzled family proteins(FZDs)andchondroitinsulfateproteoglycan4(CSPG4).Wefurtherdemonstratedthat both FZDs and CSPG4 are functional receptors for TcdB, and each can mediate binding and entryofTcdBindependently.However,howFZDsandCSPG4contributetoTcdBpathogenesis incolonictissuesduringC.difficileinfectionremainstobeestablished.Hereweproposethree aimstoaddressthiskeyknowledgebarrier. First,wefoundthatFZDsandCSPG4aredifferentiallyexpressedincolonictissues,withFZDs mainlyinthecolonicepitheliumandCSPG4largelyexpressedinsub-epithelialmyofibroblasts. We thus propose a novel ?two-receptor, two-stage? hypothesis to explain TcdB pathogenesis: TcdB first targets and enter the colonic epithelium via binding to FZDs. Disruption of the epithelium allows the toxin to gain access to sub-epithelial myofibroblasts, where TcdB utilizes CSPG4 as an alternative receptor. Aims 1 and 2 will examine this hypothesis, with Aim 1 focusing on the role of FZDs for TcdB binding and entry into the colonic epithelium during C. difficileinfectioninanimalmodels,andAim2focusingontheroleofCSPG4andmyofibroblasts in TcdB pathogenesis during C. difficile infection. Furthermore, we also found that binding of TcdB to FZDs inhibits Wnt signaling, which is essential for colonic stem cells. We thus hypothesize a novel mechanism contributing to TcdB pathogenesis: TcdB may disrupt colonic stemcellsthroughtoxin-receptorengagement,independentofitsenzymaticactivityinsidecells. Aim 3 will test this hypothesis by examining the biological consequences and pathological relevance of Wnt signaling inhibition utilizing colonic organoid models and by infection experiments in animal models with engineered C. difficile that expressing the receptor-binding domain of TcdB. We will also explore the therapeutic benefit of GSK-3 inhibitors, which modulate Wnt signaling, for reducing the damage to the colonic epithelium by TcdB during C. difficile infection. Together, these studies will provide a molecular understanding of disease progressionduringC.difficileinfectionandpotentiallyleadtonoveltherapeuticinterventions.